Biochemistry (Moscow), Vol. 64, No. 11, 1999, pp. 1219-1229. Translated from Biokhimiya, Vol. 64, No. 11, 1999, pp. 1443-1456.
Original Russian Text Copyright © 1999 by Vinogradov.
REVIEW
Mitochondrial ATP Synthase: Fifteen Years Later
A. D. Vinogradov
Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia;
fax: (7-095) 939-3955; E-mail: [email protected]
Received June 21, 1999
Abstract—Mitochondrial Fo⋅F1-H+-ATP synthase is the main enzyme responsible for the formation of ATP in aerobic
cells. An alternating binding change mechanism is now generally accepted for the operation of the enzyme. This mechanism apparently leaves no room for the participation of nucleotides and Pi other than sequential binding to (release from)
the catalytic sites. However, the kinetics of ATP hydrolysis by mitochondrial ATPase is very complex, and it is difficult to
—
explain it in terms of the alternating binding change mechanism only. Fo⋅F1 catalyzes both ∆µ
H+-dependent ATP synthesis
—
and ATP-dependent ∆µH+ generation. It is generally believed that this enzyme operates as the smallest molecular electromechanochemical reversible machine. This essay summarizes data which contradict this simple reversible mechanism and
discusses a hypothesis in which different pathways are followed for ATP hydrolysis and ATP synthesis. A model for a
reversible switch mechanism between ATP hydrolase and ATP synthase states of Fo⋅F1 is proposed.
Key words: Fo⋅F1-ATPase, ATP synthase, reversibility of enzyme catalysis, mitochondria
Gibbsian statistical mechanics may well be a fairly adequate model of what happens in the body; the picture suggested by the ordinary heat engine certainly is
not. The thermal efficiency of muscle action means next to nothing, and certainly does not mean what it appears to mean.
Norbert Weiner (“Cybernetics”, The M.I.T. press and John
Wiley and Sons, Inc., New York, London, 1961, p. 57)
1. INTRODUCTION
Fifteen years ago in the short review article
“Catalytic Properties of the Mitochondrial ATP
Synthase” published in Biokhimiya (now Biochemistry
(Moscow)) [1] I summarized experimental results
obtained in the laboratory and raised doubts about a
central dogma of enzymology: any enzyme is a catalyst
and, because of that, the molecular mechanisms of the
forward and reverse enzymatic reactions are identical;
the only difference in mechanism is the direction of the
transformation of the intermediates (microreversibility
principle). It would not be judicious to say that the
hypothesis of different reaction mechanisms for ATP
hydrolysis and synthesis catalyzed by the mitochondrial
Fo⋅F1 complex, as it was originally formulated in 1980
[2], has been agreeably accepted by the workers in the
field. Most investigators simply ignored our arguments;
others argued (explicitly or implicitly) that we attribute
a role of a Maxwell’s demon to the enzyme, thus violating the Second Law of thermodynamics.
The proton-translocating ATPases have been perhaps the most extensively studied enzymes in bioenergetics during the last decade. The outstanding achievements in the study of structure and mechanism reached
by P. Boyer and J. Walker were awarded the 1997 Nobel
Prize in Chemistry. In this essay I will summarize some
generally accepted concepts on the reaction mechanism
of the enzyme and analyze again the problem of its
reversibility. The discussion will be mostly focused on
the experimental results obtained by my own group, and
so this essay is hardly a regular review. A number of
comprehensive reviews on the subject have been published during the last decade; each of them contains hundreds references to original papers relevant to the F1ATPases studies. It is not unusual that a newcomer in
the field will find some contradictory statements in different reviews; this is especially true when the kinetic
properties of the enzyme (the particular area where our
group has some experience) are discussed. This is why
the kinetics of the enzyme is the main subject to be analyzed here.
0006-2979/99/6411-1219$22.00 ©1999 ÌÀÈÊ “Íàóêà/Interperiodica”
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VINOGRADOV
The mitochondrial H+-ATP-synthase (Fo⋅F1-H+ATPase) is an extremely complex membrane-bound
energy transducing enzyme which produces most of the
ATP present in aerobic cells. The substrates—
nucleotides and, possibly, inorganic phosphate—bind to
(release from) so-called “catalytic sites” located in the
hydrophilic part (F1); Fo, which is an intrinsic part of the
inner mitochondrial membrane, serves as the specific
proton-conducting path. It is generally accepted that the
enzyme operates as follows: the work done by the proton flow down the electrochemical potential gradient
—
(∆µ
H+) is transformed to the chemical work needed to
synthesize ATP by F1 according to Eq. (1):
ADP + Pi + n
H+out
Mg2+
+ m H → ATP + H2O + n H+in , (1)
+
where n is a quotient for stoichiometric vectorial proton
translocation from cytoplasm (H+out) to the matrix space
(H+in) and m is a stoichiometric quotient for scalar proton
consumption which is determined by the difference in
total acidity of the substrates (ADP and inorganic phosphate) and product (ATP). The exact value of n (2-4) is
not established; m is close to 1 at pH > 7.5 and depends
predominantly on pH and Mg2+ concentration, which
forms more (ATP) or less (ADP and Pi) stable complexes with the reaction product and substrates. Energydependent ATP synthesis is phenomenologically
reversible: ATP hydrolysis in tightly coupled membranous preparations results a difference between the elec—
trochemical potentials of hydrogen ions (∆µ
H+) at different sides of the coupling inner mitochondrial membrane.
Whether the ATPase activity of those preparations is a
simple reversal of ATP synthesis is not known, since the
precise mechanisms of neither ATP hydrolysis nor its
synthesis are known.
Spectacular achievements have been made in recent
years in elucidating the ternary structure of F1 at the
atomic resolution of 2.8 Å [3, 4]. The F1 (α3⋅β3⋅γ⋅δ⋅ε subunit composition) appears as a trimer of tightly packed
α⋅β-subunit pairs, and the long, rod-like γ-subunit, is
positioned in the central cavity of the overall spherical,
orange-like globular protein [3]. The ternary structure of
smaller δ- and ε-subunits and their positions relative to
the α3⋅β3⋅γ heptamer have not been revealed by crystallography. Fo has the structure a⋅b2⋅c9-12 (the minimal
composition); only the structure of the c-subunit, which
seems to be a key component of the proton-conducting
path, is well established [5]. The catalytic sites residing
on F1 are located far from the plane of the coupling
—
membrane. This spatial separation suggests that ∆µ
H+dependent ATP synthesis must be a complex electromechanochemical process. The rotational alternating
binding change mechanism originally based on quantitative measurements of isotope exchange reactions [6]
and supported by numerous studies including elegant
demonstrations of strong negative cooperativity in ATP
binding and positive cooperativity in ATP hydrolysis [7,
8] is now widely accepted [9, 10]. Essential features of
this mechanism are as follows: 1) the free energy produced by H+ flow is used not to synthesize ATP (∆G ≈
8 kcal/mole) but to release tightly bound ATP spontaneously (isoenergetically) formed at the very high-affinity ATP-binding site of the enzyme1; 2) each catalytic
site sequentially (on the turnover time scale) appears as:
empty → tight → loose in terms of occupation and tightness of nucleotide binding; 3) the occupation and mode
of nucleotide binding at one catalytic site within the α⋅β
trimer strictly determine the properties of two other
sites.
The most plausible way to realize such a mechanism
is to rotate one element of the F1 structure relative to the
trimerically arranged catalytic sites [9]. The spatial structure of F1 seems to fit ideally the proposed mechanism.
Perhaps the most impressive achievements of bioenergetic enzymology recent decades were the original prediction and further indirect demonstration [11-13] and
finally direct visualization [14, 15] of ATP-dependent
rotation of the γ-subunit (the rotor) within three α⋅βsubunit pairs structure (the stator). These exciting
results led to the conclusion that Fo⋅F1-ATP synthase is
the smallest electrical machine created by Nature and a
number of technical terms previously unknown to enzymology such as: rotor, shaft, stator, torque, clutch are
now widely used to describe the enzyme operation.
The rotational alternating binding change mechanism apparently leaves no alternative for ADP, Pi, ATP,
—
and ∆µ
H+ but to participate in the overall reaction in a
way different from that required for the substrate (product) binding (release) and driving force, respectively, as
depicted in Eq. (1). Indeed, the steady-state rates of ATP
hydrolysis or synthesis are expected to be strictly determined by occupation of the catalytic sites by nucleotides
and Pi only. However, the kinetic properties of either
soluble F1 or membrane-bound Fo⋅F1 are far from being
trivial.
Fo⋅F1-ATPase is now frequently described as a
molecular machine and the problem of its reversibility
seems to be of great importance. If, according to several language dictionaries, a general definition of a
1
This certainly does not mean that solubilized ATPase is
capable of the overall ATP synthesis. Isoenergetic ATP formation at the active site is only “half” of the enzyme
turnover. According to the proposed mechanism the driving
force for isoenergetic synthesis, at least partially, is a difference between the free energies of binding ATP and ADP +
Pi, i.e., chemical work is done by the energy released when
the protein conformation is changed from the state when it
contains ADP + Pi to the state when it contains ATP. Thus,
the isoenergetic ATP synthesis means only that the equilibrium constant for the reversible reaction ADP⋅E⋅Pi ↔ E⋅ATP
is close to 1.
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
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MITOCHONDRIAL ATP SYNTHASE
machine is an “assemblage of parts that transit forces,
motions and energy from one to another in a predetermined manner” [16] is to be accepted, the question arises: do reversible machines exist? In my opinion the
molecular machines created by Nature as well as those
created by mankind are made exclusively for unidirectional energy transduction. Thus, a special mechanism(s) that can be switched on or off by the applied
driving force, thus permitting unidirectional energy
transmission, must be a component of any machine. If
ATP synthase is a small electromechanical engine, its
performance should be described as a mechanical device
and not as thermal engine. An analysis of any mechanical machine in terms of thermodynamics seems to be
extremely complex (if not basically unsolvable) problem. Impossibility of the Maxwell demon of the molecular size realized as the well known mechanical ratchet
mechanism was brilliantly discussed in detail by R.
Feynman [17]. The key point in his arguments is that in
the long run the molecular “demon” is itself in a thermal equilibrium with a surrounding media. However,
temperature is not a parameter needed to describe a
mechanical engine, and a number of ratchet mechanisms (which are certainly not within the molecular size
range) smoothly operate in many real devices such as
the mechanical clock or windlass. I believe that thermodynamic arguments against unidirectional enzymes are
due to some misunderstanding. It is true that any
enzyme being a catalyst is not able to change the equilibrium between the substrate and product reached in a
reversible reaction. However, this is true only when the
enzyme as a mechanical device is not subjected to the
energy supply. A possible mechanism which allows an
enzyme to operate unidirectionally will be proposed
below. Another pertinent question directly related to
the present discussion can be formulated as follows: are
parts of a molecular machine (enzyme) in thermal equilibrium with the surrounding medium? If so, neither
part of this “machine” would be able to operate as an
energy transducer; it would dissipate energy (for example that of the conformational constraint) as heat, and
the device would not be a “machine”. This difficulty can
be overcome with the restriction: time needed for thermal relaxation is much longer than that needed for
“useful transport” of energy. In this case thermodynamic arguments against the unidirectional enzyme
became irrelevant simply because time is not a variable
for the thermodynamic functions which determine an
equilibrium. The lack of strong theory for an adequate
analysis of the systems composed of some parts which
are yet neither mechanical devices (cogwheel, crankshaft, etc.) nor the statistical ensemble of small particles, does not permit clear description of miniature
machines—the most conceivably perfect devices created
by Nature. The natural molecular machines fall just in
that in-between range.
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
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2. GENERAL REMARKS ON THE KINETICS
OF MITOCHONDRIAL ATPase
When soluble F1, oligomycin-sensitive purified
Fo⋅F1, or submitochondrial particles are added to a reaction mixture containing ATP and Mg2+, rapid hydrolysis of ATP is observed. The enzyme activity under “optimal” conditions corresponds to a turnover number of
several hundreds per second. It might be expected that
the standard kinetic parameters of this remarkably stable and highly active enzyme could easily be determined.
However this is not the case. Meaningful interpretation
of the enzyme kinetics is only possible when the initial
steady-state rates are measured. When the hydrolytic
activity of F1-type ATPase is considered, the initial rate,
as such, is not an unambiguous term. There are several
reasons for this. First, many preparations contain a
small natural inhibitory peptide, an ATPase inhibitor
protein (IF1) [18], which is an intrinsic part of the mitochondrial Fo⋅F1-ATP synthase. The inhibitory effect of
this protein is time-, pH-, ionic-strength-, and ATP—
dependent and may also be dependent on ∆µ
H+ and the
presence of divalent cations [18-22]. It appears that
uncontrolled contaminations of the protein inhibitor
might cause serious errors in measurements of the actual initial velocities of ATPase in many early studies,
including our own [23].
Second, the product of ATP hydrolysis, ADP, is an
inhibitor of all ATPase preparations. One type of inhibition (rapidly equilibrating competitive) [24] can be easily prevented if ATP-regenerating systems, such as phosphoenolpyruvate and pyruvate kinase or phosphocreatine and creatine kinase, are used in the assays.
However, the system becomes more complex when the
ATP-regenerating enzymes are present, and the experimental approaches are somehow restricted when
nucleotide specificity or metal requirement are being
verified because the “regenerating” enzymes themselves
depend on the nucleotides and cations (Mg2+, K+, Ca2+)
in a complex way. Another unusual type of inhibition
(modulation) by ADP which is quite different from
“simple equilibrium” and which is characteristic for all
F1-type ATPases [25, 26] and V-type ATPase [27] is a
potential source of serious errors in the ATPase assays
even in the presence of ATP-regenerating systems. For
F1-type ATPases this ADP-dependent inhibition results
in strong dependence of the measured activity on the
“history” of a particular preparation. If the enzyme is
not subjected to special treatment (see below) the ATP
hydrolytic activity is never constant and either “lag” or
“burst” are seen even in the presence of ATP-regenerating system added in “kinetic excess”. The complexities
and possible errors in measurements of the initial rates
can hardly be overestimated. Depending on particular
assay and the actual time scale, the ADP-dependent
inhibition can be easily overseen. For example, if the
1222
VINOGRADOV
“initial rates” are measured on a time scale of minutes,
the activity is proportional to a fraction of the active
enzyme which is in a slow equilibrium with completely
inactive form; this equilibrium depends on ATP, Mg2+,
and anions which are present. On the other hand, if the
time intervals are small, the apparent initial rate is again
proportional to an unknown fraction of the active
enzyme. Although the details of the unusual “hysteretic”
behavior of ATPase has been described by us about 20
years ago [28, 29] and more recently were specially
emphasized by P. Boyer [9], only a few research groups
have paid attention to this phenomenon, and thus the
results of ATPase kinetics studies should be interpreted
with great caution.
3. STEADY-STATE ATP HYDROLYSIS
Some standard kinetic parameters of ATP hydrolysis by mitochondrial ATPase are summarized in the
table and briefly discussed below.
ATP. ATP and other nucleotides (ITP, GTP) are
hydrolyzed at significant rates only in the presence of
Mg2+ or some other divalent cations (Mn2+, Co2+, Fe2+)
[30]. At high Mg2+ concentrations (2-10 mM at pH 7.08.0) the dependence of the hydrolytic rate on ATP concentration is a simple hyperbolic function within the 25000 µM range with an apparent Km of approximately
10–4 M [28]. This contrasts with several reports in the literature in which two or three Km values for ATP have
been found [31-33]. There are strong reasons to believe
that the non-hyperbolic dependence of ATPase on ATP
concentration is due to an erroneous perception of the
observed rates as the initial ones (see above). It is worth
noting that the experimentally well-documented strong
negative cooperativity in ATP binding and extremely
positive cooperativity in hydrolytic performance [7, 8]
do not conflict with the simple Michaelis–Menten kinetics of the steady-state ATP hydrolysis. So-called uni-site
catalysis is seen only under the conditions when the
enzyme concentration is several times larger than that of
ATP. The affinity for ATP binding to the “first” catalytic site is extremely high (approximately 10–12 M) and
this concentration range is never covered in the standard
steady-state kinetic measurements ([S] >> [E]t). Also,
simple Michaelis–Menten kinetics do not conflict with
the alternating binding change mechanism [6, 9]: if cooperativity of binding is very strong or absolute (as in the
“flip-flop” mechanism [34-36]), a single “entrance” for
the substrate and a single “exit” for the products would
exist.
An interesting possibility we would like to propose
is that, during the steady-state performance of F1-type
ATPase/synthase, a “gate” and special “path” operate
which determine the penetration of ATP (or ADP) from
the medium into a “catalytic cavity” where three
exchangeable sites (out of six total) are located. In the
light of the structural arrangement of F1 [3, 4], it seems
unlikely that what are assigned as “catalytic” and “noncatalytic” sites on the β- and α-subunits are readily
accessible to nucleotides from the solution. If this proposal is correct, the apparent Km for ATP is a quantitative measure of the nucleotide affinity to a “gate” and
not to the catalytic site(s) where chemical reaction takes
place; the latter would contain “occluded” nucleotides.
The Km value for ATP depends strongly on Mg2+
concentration. At high concentration (a significant
molar excess over [Mg2+]) ATP inhibits ATPase activity
[37]. This inhibition is most likely due to a decrease in
free [Mg2+], which is needed (in addition to that bound
as an ATP⋅Mg complex) for the catalysis (see below).
ADP. ADP, the product of the ATPase reaction, is
an inhibitor of the hydrolytic activity. Quantitative and
qualitative parameters for inhibition by ADP in terms of
Steady-state kinetic parameters for mitochondrial Fo⋅F1-ATPase (submitochondrial particles)*
Substrate or product
Effect on the initial rate
ATP
simple Michaelis–Menten kinetics at saturating [Mg2+]; inhibition at high concentrations when [Mg2+] is limiting
· Mg
= 1 · 10–4 M
K ATP
m
ADP
competitive inhibition
= 3 · 10–4 M
K ADP
i
Pi
no effects
Mg2+
simple Michaelis–Menten kinetics at constant [ATP⋅Mg]
Ks = (2-10) · 10–5 M,
pH-dependent
pH
very broad pH maximum at saturating [ATP⋅Mg] and free
[Mg2+]
pKa = 7.4 with limiting [Mg2+]
Apparent affinity
* The quantitative data (right column) are given for pH 7.5, 25°C, ionic strength of 0.1 M (KCl) and refer to the initial rates (10-30 sec) of ATP
hydrolysis by completely activated (preincubation with either 10 mM potassium phosphate or phosphoenolpyruvate and pyruvate kinase)
Fo⋅F1-ATPase.
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
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MITOCHONDRIAL ATP SYNTHASE
the steady-state kinetics are difficult to determine
because ADP slowly deactivates the enzyme (the term
“deactivation” will be used throughout this article to
discriminate a decrease or complete inhibition of only
the initial velocity, i.e., the lag phase in the enzyme performance, from the usual inhibition of the activity, see
below). From the initial rate measurements it has been
concluded that ADP is a simple competitive inhibitor
(with ATP) of the hydrolysis [24]. We found that free
ADP, not the ADP⋅Mg complex, competes with the
ATP⋅Mg complex for the “substrate-binding” site [37].
This finding is of particular interest because the
ADP⋅Mg complex was shown to be a true substrate for
oxidative phosphorylation catalyzed by submitochondrial particles [38] and for photophosphorylation in
chloroplasts [39]. Certainly, as it was pointed out above,
one needs to define clearly what is assumed to be the
“substrate-binding site”: one of those which are seen in
the resolved F1 structure, or it is an apparent affinity to
the entrance in the proposed “gate-specific path” mechanism which may contribute significantly in the overall
steady-state reaction.
Pi. Although Pi is equally important as ADP as a
substrate/product of ATP synthase, only a few studies,
compared with those on nucleotide binding, are available on its specific effects on the ATP hydrolysis catalyzed by F1 or by Fo⋅F1. Soluble F1 has been shown to
bind Pi with a relatively high affinity [40, 41]. It would
seem logical to expect that Pi must inhibit ATP hydrolysis with an apparent Ki similar to its Km value for oxidative phosphorylation (in the millimolar range [38]).
However, this is not the case, and Pi either has no effect
on ATP hydrolysis or it slightly stimulates ATPase
activity, as do several other anions. It is extremely difficult, if not impossible, to construct a kinetic scheme
which includes the reversible Pi release step and which
does not contain a Pi inhibition term in an equation for
the steady-state reaction rate. This problem has been
somehow overlooked in many detailed discussions of the
enzyme mechanism. The only possible explanation for
the absence of its inhibitory effect is that Pi irreversibly
dissociates from the active site during ATP hydrolysis:
this proposal immediately creates an unavoidable problem in viewing ATP synthase as a reversible ATPase.
Mg2+. It is well established that Mg2+ (or some other
divalent cation) is needed for ATPase activity [23, 30,
42]. Since the early publications on ATPase kinetics [23,
42], statements on the inhibition of ATPase by free
excess Mg2+ have been widely circulated in the literature.
Recently, we have reinvestigated the problem and found
no inhibition of the enzyme activity by Mg2+ (up to 1015 mM) provided that true initial rates are measured. It
appears that the previously reported inhibitory effects of
Mg2+ were due to underestimations of its other effect:
Mg2+-induced, ADP-dependent (or vice versa) slow
deactivation of the enzyme. Moreover, we found that
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
1999
1223
free Mg2+, in addition to that bound in the true substrate
(ATP⋅Mg)2–, is needed for ATPase activity [37]. The
kinetic analysis suggests that activating Mg2+ is involved
in the overall reaction according to the so-called “pingpong” mechanism, i.e., it participates after irreversible
dissociation of one of the products from the active site.
Since the structure of α3⋅β3⋅γ at 2.8 Å resolution is now
available [3, 4], an obvious question arises: where the
Mg2+-specific “activating” site is located? The structures
of the nucleotide-binding sites in asymmetric F1 containing Mg2+–nucleotide complexes bound to the α and
β subunits were visualized. Surprisingly, no Mg2+ other
than that originating from the Mg2+–nucleotide complexes was seen, despite the presence of adenosine-5'phosphoimidophosphate, ADP, Mg2+, and sodium azide
and the absence of Pi in the crystallization medium [43].
It is possible that the Mg2+-specific site is located on the
disordered “invisible” part of the γ-subunit.
When only a limiting concentration of free Mg2+ is
available, ATPase activity becomes strongly pHdependent, as to be expected if Mg2+ binds to a deprotonated group with a pKa of 7.4 [37].
∆—
µ H+. The ATPase activity of intact mitochondria
is greatly stimulated by uncoupling agents. Unfortunately, ATPase of coupled submitochondrial particles is
only slightly (not more than twofold) accelerated when
—
∆µ
H+ is collapsed. It should be admitted that our knowl—
edge on the possible effects of ∆µ
H+ on steady-state ATP
hydrolysis are very limited.
The kinetic data summarized in the table suggest
the simplest kinetic scheme for the steady-state ATPase
reaction as depicted in Fig. 1. The initial steady-state
rate reaction mechanism (under the conditions where
slow deactivation of the enzyme is prevented) are shown
as the sequence of steps (1)-(5). In addition to what was
discussed above, some other comments concerning the
reaction sequence is worth mentioning. The participation of free Mg2+ corresponds to so-called ping-pong
kinetics. Mechanistically this means that “activating”
Mg2+ participate in the reaction after release of one of
the products (ADP or Pi). We believe that Pi leaves the
active site before ADP. It is known that Pi is not
required for binding of ADP by the enzyme. Also the
absence of any inhibitory effect of Pi on Fo⋅F1-ATPase
suggests that it leaves the enzyme during uncoupled
ATP hydrolysis from a site with very low or no affinity
and this is taken as the thermodynamic reason for the
kinetic sequence of the products release. The simple
competitive inhibition of ATPase by free ADP is in
accord with the reaction sequence depicted in Fig. 1,
where free ADP leaves the catalytic site via a Mg2+dependent mechanism. If the proposal for γ-subunit
Mg2+-binding is correct, the structural arrangements of
the events shown as the reaction scheme in Fig. 1 would
be as follows: free ADP, that is left at the catalytic site
after the departure of Pi⋅Mg, is then released to the solu-
1224
VINOGRADOV
tion via an interaction with the Mg2+-liganded arm of γsubunit. It should be emphasized that the reaction
scheme is based an well established fact: the steady-state
ATP hydrolysis is described by the simple Michaelis–
Menten kinetics [28] as it would be expected if a single
substrate-binding site participate in the reaction mechanism. As it was pointed out before, the same behavior is
expected if two or more sites participate and high or
absolute cooperativity in the catalytic performance exist.
The final note is that the proposed kinetic scheme is not
aimed to describe the structural rearrangements during
the reaction. For example, the release of free ADP by
the “magnesium arm” may correspond to the key point
of the rotational mechanism with direct interaction
between the ADP-containing α⋅β-subunit and γ-subunit
which sequentially serves α⋅β pairs.
4. NON-STEADY-STATE ENZYME KINETICS
Effect of ADP. Mitchell and Moyle were the first to
observe a relatively slow time-dependence of some kinetic properties of the ATPase: inhibition by Mg2+ and activation by some anions (sulfite) [44, 45]. This phenome(2)
Å · (Ò · Ì)
Å · (D · Ì · Pi)
(1)
(3)
Ò·Ì
M + Pi
Fast
D
Å
Å · (D)
?
T
Ma + D
(5)
(4)
Å · (Ìà)(D)
Ma
(6)
(8)
Slow
HSO3–
Slow
Å* · (Ìà)(D)
(7)
(N3–)
µ H+
∆—
ATP synthase
(N3–) · Å* · (Ìà)(D)
Fig. 1. Kinetic model of ATP hydrolysis by the mitochondrial ATPase. Reactions (1)-(5) describe the initial steady-state hydrolysis. T,
D, M, and Ma stand for ATP, ADP, and Mg2+ bound to nucleotide and free (activating) Mg2+, respectively. Step (5) is shown as an irreversible reaction because for the initial rate conditions [D] = 0, whereas the irreversibility of step (3) is due to the properties of the catalytic site. The participation of Ma is shown as two separate steps—association, step (4) (pH-dependent binding with pKa equal to 7.5
[37, 50, 71]), and dissociation, step (5)—for the sake of clarity; the kinetic behavior will be the same if Ma would be permanently bound
to one of the enzyme subunits. Steps (6)-(8) describe the slow changes of the enzyme activity (see Section 4). The large curly bracket indicates the intermediates which are subjected to the energy-dependent transformation (see Section 5). The step-by-step mechanism of the
ATP-dependent activation of the ADP-deactivated enzyme (8) remains unknown.
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
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MITOCHONDRIAL ATP SYNTHASE
non, although demonstrated for ATPases from a variety
of sources [44-47], was somehow ignored for a long time.
In 1979, it was discovered in my group that ADP is
responsible for the slow transient kinetics of ATPase
[25]. Since then, a number of detailed studies have
shown that an ADP(Mg2+)-dependent slow active/inactive transition is a universal property of F1-type
ATPases. Some essential features of this still enigmatic
phenomenon are briefly summarized below.
1. When ATP at relatively low concentrations
(~Km) is hydrolyzed by ATPase free of protein inhibitor
in the absence of an ATP-regenerating system, a rapid
decline in the activity is observed which is not due to a
decrease in the substrate concentration. Further addition of the enzyme results in a newly observed activity
which would be expected if the enzyme added previously have been irreversibly inactivated [25].
2. Brief preincubation of Fo⋅F1 (submitochondrial
particles) or soluble F1 with very low, almost stoichiometric concentrations of ADP in the presence of Mg2+
results in a considerable lag (in a time scale of minutes)
in the onset of ATPase activity measured in the presence
of an ATP-regenerating system. Qualitatively the same
phenomenon is seen for standard F1 preparations after
preincubation with Mg2+ alone, but it is absent when
nucleotide-depleted F1 is assayed with an ATP-regenerating system after preincubation with Mg2+ [29, 48].
3. The initial rates of ATP hydrolysis by submitochondrial particles or F1 measured under unified conditions (with or without regenerating enzymes) are poorly
reproducible. The activities are markedly increased and
becomes quite reproducible after prolonged preincubation of the preparations in the presence of phosphoenolpyruvate plus pyruvate kinase or phosphocreatine
and creatine kinase. After such activation, the ATPase
activity measured with an ATP-regenerating system
becomes biphasic: a rapid initial phase slowly decreases
to 50-70% of the original level, and the reaction then
proceeds at a constant rate [29].
4. Neither azide (a specific inhibitor of F1-ATPase)
nor sulfite (activator of the enzyme) affect the initial
rapid phase of ATP hydrolysis, whereas the delayed
steady-state rate is inhibited by azide and stimulated by
sulfite [49]. Hydrolysis of other nucleotides (ITP, GTP)
by activated ATPase occurs at a constant rate (no “lags”
or “bursts”) and is insensitive to azide or sulfite.
Preincubation of the enzyme with IDP or GDP has no
effect on its ATPase activity, whereas preincubation
with ADP completely blocks the initial rates of hydrolysis of the other nucleotides.
5. The duration of the lag-phase in the ATP regeneration assay depends on the ATP concentration
approximately in the same way as does the steady-state
ATPase activity itself. Thus, activation of the enzyme
during preincubation with pyruvate kinase plus phosphoenolpyruvate (no ATP is present, see point 3 above)
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
1999
1225
occurs much more slowly than in the assay system in the
presence of added ATP.
6. When ATPase is first deactivated by ADP +
Mg2+ and then passed through a Sephadex column in the
presence of EDTA to remove Mg2+, the enzyme becomes
“active” and can be deactivated again by added Mg2+
[50]. In other words, Fo⋅F1 bears a specific site for Mg2+
binding with an apparent affinity close to that measured
in the steady-state kinetics experiments. Mg2+-affinity as
revealed by these two independent approaches is strongly pH-dependent. It increases at alkaline pH and
decreases upon acidification [37, 50].
7. Pi, which does not affect ATP hydrolysis, strongly modulates the ADP(Mg2+)-induced deactivation. It
decreases the affinity for ADP at the deactivating site, so
that the enzyme can be reactivated equally well either by
pyruvate kinase or by Pi [51]. Pi also significantly
increases the rate of EDTA-induced activation (see
point 6 above) [50].
The results listed above suggest, that in addition to
the rapid ATP hydrolytic steady-state turnover route
(Fig. 1, steps (1)-(5)), an additional pathway of the
enzyme transformation exists (as described in Fig. 1 by
steps (6), (7), and (8)), which explains the pre-steadystate kinetics of ATP hydrolysis and also the inhibitory
and activating effects of azide and sulfite.
5. UNIDIRECTIONAL TRANSFORMATIONS
OF ATPase/SYNTHASE AND REVERSIBILITY
OF THE ENZYME OPERATION
ADP dissociates from its ATPase inhibiting site
very slowly (the first-order rate constant varies from 0.1
to 1 min–1). Thus, this site can not be considered as one
which participates in the rapid ATPase reaction. Many
years ago we showed that ADP(Mg2+)-deactivated
ATPase is able to catalyze oxidative phosphorylation
without any lag in ATP formation [2]. This observation
led us to conclude [1, 52, 53] that, when F1 catalyzes
—
ATP hydrolysis or ∆µ
H+-dependent ATP synthesis, the
enzyme exists in at least two slowly interconvertible conformations. In other words, the microreversibility principle, which is usually quantitatively expressed in enzymology as Haldane relationships does not hold for F1ATPase. The main difficulty in obtaining experimental
evidence for or against such a proposal is that the conditions for measurement of the reaction in the two directions are quite different. Some recent findings relevant
to this problem are briefly discussed below.
The ADP(Mg2+)-deactivated enzyme can be
trapped by azide [49] (see Fig. 1, step (7)). Surprisingly,
tightly coupled submitochondrial particles in which
ADP-deactivated F1 is trapped by azide catalyze succinate-supported ATP synthesis at the same rate as control samples, whereas both uncoupled ATPase activity
1226
VINOGRADOV
—
and ATP-dependent ∆µ
H+-generation ceased [53].
Further studies of this unusual unidirectional inhibition
—
have revealed that ∆µ
H+ (“substrate” or “product” of the
reactions) is a strong effector of the Fo⋅F1 complex [54].
When ATP synthesis catalyzed by the azide-trapped
deactivated enzyme is stopped because of the collapse of
—
∆µ
H+, no further hydrolysis of added ATP proceeds.
However, if ATP is added first, rapid hydrolysis
becomes evident after subsequent addition of an uncoupler to the submitochondrial particles, and the reaction
decreased slowly over time because of inhibition by
—
azide. It has been shown that this striking effect of ∆µ
H+
is not due to back pressure (inhibition by a “product”),
—
and the only ligand that prevents ∆µ
H+-dependent
enzyme transformation is free ADP. Our interpretation
of these results is shown in Fig. 2 [54]. The scheme does
—
not describe the detailed mechanism of ∆µ
H+-dependent
transformation of the enzyme. The key point is that the
transition ATP hydrolase → ATP synthase is energyand ADP-dependent. We observed in our preliminary
—
experiments (unpublished) that the ∆µ
H+ value needed to
transform the enzyme is considerably less than that
which is needed as a driving force for ATP synthesis.
The “difficult” point in the scheme is that ADP catalyzes
—
the ∆µ
H+-dependent transition. It may be speculated that
this “catalysis” is due to ADP binding at one site and to
—
its ∆µ
H+-dependent release from another site.
—
A similar (if not identical) phenomenon of ∆µ
H+dependent transformation of ATPase in chloroplasts has
been well documented [55-59], and it was interpreted
using quite complex kinetic schemes where the coupled
ATPase is considered as a “reversible” machine [57].
More recently, our results on the unidirectional inhibition of ATPase activity by azide has been confirmed in
the elegant studies on the reconstituted system com-
Active ATP
hydrolase
posed of Thermus thermophilus Fo⋅F1-ATP synthase and
bacterial rhodopsin [60].
The different reaction pathways for ATP synthesis
and hydrolysis catalyzed by Fo⋅F1 has been discussed in
our previous publications using a language for enzyme
kinetics which is not enjoyably acceptable by most
scholars in the field. The remarkable progress in structural studies of the enzyme [3, 4] enable us to present our
working hypothesis as the scheme shown in Fig. 3. The
key point in this scheme is that the “clutch” between a
rotating γ-subunit and α3⋅β3 complex is changed when
the enzyme operates in different directions. The simplest
driving mechanism to shift the “clutch” from one mode
of operation to the other could be the vertical (perpen—
dicular to the plane of the membrane) ∆µ
H+-dependent
movement of one or several subunits of Fo that are firmly attached to the F1 part. The minimal requirement for
such a movement would be the positioning of a charged
(or potentially charged) group within the membrane—
embedded area to function as a ∆µ
H+-sensitive sensor.
This model is different from those in which elastic spring
(γ-subunit) is proposed for the transmission of a torque
from Fo to the catalytic sites of F1 [15, 61]. I believe that
a rigid ratchet mechanism operates in the electromechanical energy transduction. The difficulties which
arise when “conformational” energy must be “transported” (which is equivalent to the energy storage in a
form of the elastic Young module) were briefly discussed
above (see “Introduction”). In my opinion, if Nature has
created an electromechanical motor, it has also provided it with a special mechanism to allow it to work effectively as a mechanoelectrical generator, just like similar
devices created by mankind are differently constructed
although they use the same physical principles when
they operate in opposite directions.
Deactivated ATP
hydrolase
Active ATP
synthase
ADP
H
Å •• Mg
ADP
H
ADP
•
Å • Mg
S
+ ∆µ H+
—
Å •• Mg
Uncoupler
–∆—
µ H+
—
Fig. 2. ∆µ
H+-dependent transformation of the catalytic activities of Fo⋅F1 complex. See text for explanations.
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
1999
MITOCHONDRIAL ATP SYNTHASE
1
1227
2
3
ADP + Pi + H+
AÒP
ADP + Pi + H+
AÒP
ADP + Pi + H+
AÒP
α·β
α·β
γ
α·β
→
nH+
δ·ε
C12
a·b
α·β
γ
→
(–)
AH
α·β
α·β
nH+
—
AÒP ± ∆µ H+
C12
δ·ε
a·b
–
ADP ± ∆µ H+
—
AH
C12
(+)
a·b
→
→
nH
nH+
H
N
δ·ε
A–
+
+
S
Fig. 3. The hypothetical energy-dependent switch-mechanism for Fo⋅F1-ATP hydrolase/synthase. For the sake of simplicity Fo⋅F1 complex is shown as being composed of three parts. The catalytic part is shown as only one (out of three total) working pair of the α⋅β-subunits (shadowed). The drive is shown as the proton channel (subunits c) and the γ-subunit. The third part is a switch-device which is
shown as the combination of a-, b-, δ-, and ε-subunits. The spatial organization of the subunits is not specified. For detailed information on the structure and dynamics of separate subunits the reader is addressed to an excellent mini-review series published in the Journal
—
of Bioenergetics and Biomembranes [72]. An acidic group AH is shown as an ∆µ
H+ sensor; a similar model can be easily constructed with
a positively charged group. The enzyme state shown in the center corresponds to inactive (incapable of neither hydrolysis nor synthesis)
form (N, neutral) in which the proton channel is closed. The synthase state (S) and the hydrolase state (H) are shown on the right and
the left, respectively. The different “conformations” may corresponds to the “collapsed” and “extended” states of Fo⋅F1 which were
described by Green’s group many years ago [73]. It is assumed that both S and H states are metastable. Nucleotides participating in the
switch mechanism are shown in italic to emphasize their regulatory function. It does not seem to be inconceivable that the binding sites
(or type of binding) which are considered as “noncatalytic” sites for ATP hydrolysis operate as “catalytic” sites in the ATP synthesis
reaction.
What are the biological advantages of an enzyme
operating differently in forward and reverse directions?
In a broad sense the answer can be formulated as follows: the separation of the forward and reverse mechanisms is required for improvement of energy transduction efficiency. The term “efficiency” is used here not to
mean the ordinary thermal efficiency. It is very hard, if
not impossible, to define what is useful for mitochondrion, cell, or whole organism. The quantitative comparison of the thermal efficiencies for some ATP-producing
metabolic pathways, such as glycolysis and oxidative
phosphorylation, which are often circulated in many
biochemistry textbooks, seems to me rather naive. In my
understanding the meaning of an “efficiency” is that
mitochondria provide the physiologically required rate
of ATP synthesis independent of Lipman’s phosphoryl
potential [62]. On the other hand, an efficient molecular
—
machine must maintain ∆µ
H+ across the mitochondrial
membrane when “glycolytic” ATP is to be used when
BIOCHEMISTRY (Moscow) Vol. 64 No. 11
1999
needed, again independent of phosphoryl potential. The
latter appears to occur only under extreme conditions, at
least for mammalian cells. An “efficient” ATP synthase
should also provide safe electric insulation during its
operation. As discussed elsewhere [63, 64], any energytransducing membrane-bound enzyme is potentially
“dangerous” for the others because it contains a proton
conducting path. Finally, an efficient machine must be
under precise metabolic control. The different mechanisms for forward and reverse reactions make independent metabolic control of the enzyme activities possible,
since different conformations of the protein are likely to
be susceptible in different ways to the same (or different)
ligands. It is well known that different anabolic and
catabolic pathways are widely exploited for metabolic
regulation in cells. Different enzymes, for example,
phosphofructokinase and fructose-bis-phosphatase,
operate in glycolysis and gluconeogenesis; again isoenzymes, such as different lactate dehydrogenases, partici-
1228
VINOGRADOV
pate in the metabolism of the same substrates in different organs or cell compartments. The rapidly expanding
concept that considers an enzyme as a molecular
machine makes me think that “the one-way traffic” principal is also realized at the level of a single enzyme.
Several recent findings indicate that such a principal
applies to another mitochondrial molecular machine,
i.e., Complex I of the mitochondrial respiratory chain
[64, 65].
6. CONCLUSIONS AND PERSPECTIVES
Tremendous experimental effort has been made for
more than half of a century to determine the molecular
mechanism of ATP synthesis during oxidative phosphorylation and photophosphorylation. One recent review
on the subject contains 284 references, citing almost
exclusively papers published since 1990 [66] ! In spite of
spectacular progress in the field, it can not be denied
that this problem is far from being solved. The great
majority of mechanistic studies in the past have been
focused on the ATP hydrolysis reaction catalyzed by F1
which, by definition, is only a hydrolase. An obvious
reason for that is that the “synthetic” reaction is far
—
more complex: the ∆µ
H+-generating system must be
included in any assay. Does it mean that we are looking
for the lost object exclusively within the illuminated
area? Most studies have been performed with explicit or
implicit assumption that the enzyme reversibly operates
—
within the Fo⋅F1 ∆µ
H+-dependent ATP synthase complex.
I believe that substantial data have been accumulated in recent years which hardly fit this simple, perhaps
unintentionally desired, assumption. Hopefully, in the
future, more emphasis will be focused on Fo⋅F1 operating in the “physiological direction” (at least for the
mammalian enzyme). One may expected that many current dogmas concerning the enzyme mechanism will be
reevaluated. After brilliant experiments, where rotation
of the γ-subunit within trimeric α⋅β pairs composed F1,
was directly visualized [14, 15], the rotary mechanism of
F1 operation comes to be generally accepted. However,
it does not seem improbable that the rotation does not
occur at all when the complete Fo⋅F1 complex operates
—
—
as a ∆µ
H+-consuming or ∆µ H+-generating device. If some
analogy with mechanical machines is to be used, many
examples can be found when some part moves reciprocally in the assembled mechanism and it starts to rotate
when the device is disassembled. It is hard to predict
when the structure of Fo⋅F1 at an atomic resolution will
be available. Other techniques such as modern cryo-elec—
tron microscopy [67, 68] may reveal the ∆µ
H+-dependent
difference in the structure of Fo⋅F1 as predicted by the
model shown in Fig. 2. Another prediction of the model
is that the reactivities and the sites of incorporation for
some irreversible inhibitors (nucleotide analogs) are
—
expected to be ∆µ
H+-dependent. Perhaps the most direct
proof of our hypothesis would be construction of sitedirected mutants capable of coupled ATPase and incapable of oxidative phosphorylation (or vise versa). In
fact, a recent report on the α-subunit mutated thermophilic F1 [69] strongly support our hypothesis: the
mutant catalyzes only the initial rapid phase of ATP
hydrolysis (see point 3 in Section 4), whereas this mutated F1 incorporated (together with bacteriorhodopsin
and Fo) into proteoliposomes is perfectly capable of
light-induced ATP synthesis [60]!
Enzymatic catalysis is so complex that it is hard to
expect formulation of the precise mechanism for any
enzyme: the answer concerning a mechanism depends on
what particular question is asked. The enzymes are
superb because of their catalytic efficiency, specificity
and, perhaps most importantly, because of their
extremely refined control mechanisms. The later aspect
of ATP synthase operation is still at a primitive stage. It
hard to believe that “The ATP synthase – a splendid
molecular machine” [70] is not subjected to sophisticated metabolic control.
The work carried out in my laboratory was partially supported by the Russian Foundation for Basic
Research (Grants 96-04-48185, 99-04-48082) and by the
Program for Advanced Schools in Science (Grant 96-1597822). I thank all my colleagues past and present for
many stimulating discussions. The great help of Mr. F.
Kasparinsky and Dr. V. Grivennikova in the preparation of the manuscript is gratefully acknowledged.
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